carnosol and Colonic-Neoplasms

Cytoprotective effects by upregulating cellular expression levels of antioxidant proteins are attributed to a significant number of food ingredients. Evaluation of those cytoprotective effects and identification of the most active components requires reliable and comprehensive proteomic strategies. Thus, promising potential bioactive ingredients could be determined for the prevention of various diseases, including colon cancer formation. We established a multiplexed nanoLC-MS/MS targeted proteomic method, operated in scheduled selected reaction monitoring mode (sSRM), to record modulation of the expression levels of six major antioxidant proteins induced by dietary phytochemicals. Relative quantification was achieved by isotope-coded protein labels (ICPLs) and based on two to three proteotypic peptides per target protein. The assay provided accurate (mean relative error 6.4%) and precise (mean RSD 7.4%) quantification. Incubation experiments were carried out in primary human colon epithelial cells (HCoEpiCs) and revealed significant upregulation of NAD(P)H dehydrogenase [quinone] 1 (up to threefold) and thioredoxin reductase 1 (up to twofold) by 10μM sulforaphane (from broccoli), 5μM carnosol (rosemary), and 20μM cinnamaldehyde (cinnamon). The latter two substances additionally upregulated heme oxygenase-1 and were identified as the most active components in the test set. The results provide additional evidence for nutritive cytoprotection in human colon cells.. Targeted proteome analysis using LC coupled to scheduled selected reaction monitoring (sSRM)-MS is a highly flexible and reliable method to monitor protein expression profiles. The present study screened modulators occurring in food, which may be protective against colon cancer by inducing cytoprotective enzymes. Primary human colonic epithelial cells were used because they model the conditions in healthy gut tissue better than immortalized cells. Thus, an LC-MS/MS-sSRM protocol was established and validated including relative quantification of cytoprotective protein expression by isotope-coded protein labels, because metabolic labelling cannot be applied for primary cells. The present study demonstrated that the major components of cinnamon and rosemary, respectively, i.e. cinnamaldehyde and carnosol, are potent alimentary candidates to increase the expression of cytoprotective enzymes in the human colon. Among the investigated enzymes, NAD(P)H dehydrogenase [quinone] 1 (NQO1) was most susceptible towards modulation by phytochemicals. NQO1 exerts its cytoprotective activity by detoxifying electrophilic and oxidative xenobiotics with quinone structure.

Topics: Abietanes; Acrolein; Cells, Cultured; Colon; Colonic Neoplasms; Cytoprotection; Epithelial Cells; Humans; Isotope Labeling; NAD(P)H Dehydrogenase (Quinone); Phytochemicals; Proteomics; Tandem Mass Spectrometry; Thioredoxin Reductase 1

Carnosic acid (CA) and carnosol (CS) are two structurally related diterpenes present in rosemary herb (Rosmarinus officinalis). Although several studies have demonstrated that both diterpenes can scavenge free radicals and interfere in cellular processes such as cell proliferation, they may not necessarily exert the same effects at the molecular level. In this work, a shotgun proteomics study based on stable isotope dimethyl labeling (DML) and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) has been performed to identify the relative changes in proteins and to gain some light on the specific molecular targets and mechanisms of action of CA and CS in HT-29 colon cancer cells. Protein profiles revealed that CA and CS induce different Nrf2-mediated response. Furthermore, examination of our data revealed that each diterpene affects protein homeostasis by different mechanisms. CA treatment induces the expression of proteins involved in the unfolded protein response in a concentration dependent manner reflecting ER stress, whereas CS directly inhibits chymotrypsin-like activity of the 20S proteasome. In conclusion, the unbiased proteomics-wide method applied in the present study has demonstrated to be a powerful tool to reveal differences on the mechanisms of action of two related bioactive compounds in the same biological model.

Topics: Abietanes; Chromatography, Liquid; Colonic Neoplasms; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Homeostasis; HT29 Cells; Humans; Isotope Labeling; Proteasome Endopeptidase Complex; Proteomics; Signal Transduction; Tandem Mass Spectrometry; Unfolded Protein Response

Topics: Abietanes; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Chromatography, Liquid; Colonic Neoplasms; Gas Chromatography-Mass Spectrometry; HCT116 Cells; HT29 Cells; Humans; Mass Spectrometry; Picrates; Plant Extracts; Rosmarinus

Carnosol, an active constituent of rosemary, has been reported to possess anti-inflammatory and anticancer activities. However, the molecular mechanisms underlying the anticancer effects of carnosol remain poorly understood. In the present study, we found that carnosol significantly reduced the viability of human colon cancer (HCT116) cells in a concentration- and time-dependent manner. Treatment of cells with carnosol induced apoptosis, which was associated with activation of caspase-9 and -3 and the cleavage of poly-(ADP-ribose) polymerase (PARP). Incubation with carnosol elevated the expression of Bax and inhibited the levels of Bcl-2 and Bcl-xl. Carnosol induced expression of p53 and inhibited that of murine-double minute-2 (Mdm2). Moreover, carnosol generated reactive oxygen species (ROS), and pretreatment with N-acetyl cysteine abrogated carnosol-induced cleavage of caspase-3 and PARP. The constitutive phosphorylation, the DNA binding and reporter gene activity of signal transducer and activator of transcription-3 (STAT3) was diminished by treatment with carnosol. To further elucidate the molecular mechanisms of STAT3 inactivation, we found that carnosol attenuated the phosphorylation of Janus-activated kinase-2 (Jak2) and Src kinase. Pharmacological inhibition of Jak2 and Src inhibited STAT3 phosphorylation. Furthermore, carnosol attenuated the expression of STAT3 target gene products, such as survivin, cyclin-D1, -D2, and -D3. Taken together, our study provides the first report that carnosol induced apoptosis in HCT116 cells via generation of ROS, induction of p53, activation of caspases and inhibition of STAT3 signaling pathway.

Topics: Abietanes; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cyclin D1; Cyclin D2; Cyclin D3; DNA-Binding Proteins; HCT116 Cells; Humans; Inhibitor of Apoptosis Proteins; Janus Kinase 2; Phosphorylation; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-mdm2; Reactive Oxygen Species; Signal Transduction; src-Family Kinases; STAT3 Transcription Factor; Survivin; Tumor Suppressor Protein p53

Carnosol, a constituent of the herb, rosemary, has shown beneficial medicinal and antitumor effects. Using the C57BL/6J/Min/+ (Min/+) mouse, a model of colonic tumorigenesis, we found that dietary administration of 0.1% carnosol decreased intestinal tumor multiplicity by 46%. Previous studies showed that tumor formation in the Min/+ mouse was associated with alterations in the adherens junctions, including an increased expression of tyrosine-phosphorylated beta-catenin, dissociation of beta-catenin from E-cadherin, and strongly reduced amounts of E-cadherin located at lateral plasma membranes of histologically normal enterocytes. Here, we confirm these findings and show that treatment of Min/+ intestinal tissue with carnosol restored both E-cadherin and beta-catenin to these enterocyte membranes, yielding a phenotype similar to that of the Apc(+/+) wild-type (WT) littermate. Moreover, treatment of WT intestine with the phosphatase inhibitor, pervanadate, removed E-cadherin and beta-catenin from the lateral membranes of enterocytes, mimicking the appearance of the Min/+ tissue. Pretreatment of WT tissue with carnosol inhibited the pervanadate-inducible expression of tyrosine-phosphorylated beta-catenin. Thus, the Apc(Min) allele produces adhesion defects that involve up-regulated expression of tyrosine-phosphorylated proteins, including beta-catenin. Moreover, these data suggest that carnosol prevents Apc-associated intestinal tumorigenesis, potentially via its ability to enhance E-cadherin-mediated adhesion and suppress beta-catenin tyrosine phosphorylation.

Topics: Abietanes; Adenoma; Animals; beta Catenin; Cadherins; Cell Adhesion; Cell Membrane; Colonic Neoplasms; Cytoskeletal Proteins; Enterocytes; Female; Intestine, Small; Mice; Mice, Inbred C57BL; Phenanthrenes; Phosphorylation; Rosmarinus; Trans-Activators; Tyrosine; Vanadates